Antigenotoxic role of Centella asiatica L. extract against cyproterone acetate induced genotoxic damage in cultured human lymphocytes

Available online at www.sciencedirect.com

Toxicology in Vitro 22 (2008) 10–17 www.elsevier.com/locate/toxinvit

Antigenotoxic role of Centella asiatica L. extract against cyproterone acetate induced genotoxic damage in cultured human lymphocytes Yasir Hasan Siddique a,*, Gulshan Ara a, Tanveer Beg a, Mohammad Faisal b, Mukhtar Ahmad b, Mohammad Afzal a a

Human Genetics and Toxicology Laboratory, Section of Genetics, Department of Zoology, Faculty of Life Sciences, Aligarh Muslim University, Aligarh 202 002, UP, India b Forest Entomology Division, Forest Research Institute, Dehradun 248 006, UA, India Received 14 February 2007; accepted 5 July 2007 Available online 18 July 2007

Abstract The majority of the Indian population use traditional natural preparations derived from plant material for the treatment of various diseases, and for that reason it becomes necessary to assess the mutagenic potential or modulating action of plants extract when associated with other substances. The genotoxicity testing provides human a risk assessment. Earlier in vitro and in vivo studies reveal that the plant extracts from various parts of the plant play a modulating role in xenobiotic effects. Identification and characterization of some active principles may lead to the development of the strategies to reduce the risk for developing cancer in humans. Cyproterone acetate (CPA), a synthetic progestin is not only a genotoxic agent but also a tumor initiating agent. It is used in oral contraceptives formulations and also in the treatment of various sexual and metabolic disorders. In this context, the antigenotoxic effect of Centella asiatica L. extract was studied against the genotoxic effect induced by CPA on human lymphocytes using chromosomal aberrations and sister chromatid exchanges as parameters. The treatment of the two doses of CPA, i.e. 20 and 30 lM was given along with the C. asiatica extract at the dosages of 1.075 · 10 4, 2.125 · 10 4, 3.15 · 10 4 and 4.17 · 0 4 g/ml of culture medium. A clear dose dependent decrease in the genotoxic damage of CPA was observed, suggesting a protective role of C. asiatica extract during CPA therapy. The results of the present study suggest that the plant extract per se do not have genotoxic potential, but can modulate the genotoxicity of CPA on human lymphocytes in vitro.  2007 Elsevier Ltd. All rights reserved. Keywords: Centella asiatica; Cyproterone acetate; Human lymphocytes; Chromosomal aberrations; Sister chromatid exchange

1. Introduction Centella asiatica belongs to the family Umbelliferae. It is also known as mandukparni or Indian Penny Wort. It is found in swampy area of India, commonly found as a weed crop fields and other waste places throughout India upto an altitude of 600 m (Dastur, 1962). The crude extract of

*

Corresponding author. E-mail address: yasir_hasansiddique@rediffmail.com (Y.H. Siddique).

0887-2333/$ - see front matter  2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2007.07.001

C. asiatica and the products derived from glycoside were used as oral antifertility agents (Dutta and Basu, 1968). The extract of C. asiatica extract possesses antioxidant (Gupta and Flora, 2006), anti-inflammatory (Guo et al., 2004), immunomodulating (Punuree et al., 2005), antitumor (Babu et al., 1995), antiproliferative (Yoshida et al., 2005), radioprotective (Sharma and Sharma, 2002) and antigenotoxic (Siddique et al., 2007) properties. The extract of C. asiatica L. has certain bioactive terpene acids such as asiatic acid, madecassic acid and their respective glycoside, asiaticoside and madecassoside (Inamdar et al., 1996).

Y.H. Siddique et al. / Toxicology in Vitro 22 (2008) 10–17

Asiatic acid decreased the viability and induced apoptosis in human melanoma SK-MEL-2 cells (Park et al., 2005). Asiaticoside and madecassoside have anti-psoriatic properties (Sampson et al., 2001). Asiaticoside, has wound healing activity (Shukla et al., 1999), promotes fibroblast proliferation (Lu et al., 2004) and increases the level of enzymatic and non-enzymatic antioxidants (Shukla et al., 1999). There are some phenolic compounds in the extract of C. asiatica, having the activity same as that of the a-tocopherol (Zainol et al., 2003). The crude extract of C. asiatica was shown to be non-toxic in normal human lymphocytes (Babu et al., 1995) and reduced the genotoxic effects of methyl methanesulphonate and cyclophosphamide in cultured human lymphocytes (Siddique et al., 2007). Synthetic progestins are used in the treatment of sexual and metabolic disorders, and also in oral contraceptives (IARC, 1999). Prolonged use of oral contraceptives has been shown to develop various types of malignancies in human and experimental animals (IARC, 1999). Earlier studies reveal that synthetic progestins have DNA damaging potential (Joosten et al., 2004; Siddique and Afzal, 2004a; Siddique and Afzal, 2004b). The genotoxic effects of synthetic progestins can be reduced by the use of antioxidants (Siddique et al., 2006; Siddique et al., 2005; Ahmad et al., 2002) and natural plants products (Siddique and Afzal, 2005a; Siddique et al., 2006; Siddique et al., in press). Cyproterone acetate (CPA) is a tumor initiating agent in the liver of female rats (Deml et al., 1993; Martelli et al., 1996). It induced micronucleus in rat liver cells (Martelli et al., 1996), chromosomal aberrations in V79 cells (Kasper et al., 1995), and human peripheral blood lymphocytes (Reimann et al., 1996; Siddique and Afzal, 2005b), and also sister chromatid exchanges in human peripheral blood lymphocytes in vitro (Siddique and Afzal, 2005b). Studies of genotoxicity and antigenotoxicity of natural plant extracts can help us to evaluate the safety and effectiveness of herbal health products (Romero-Jimenez et al., 2005). The herbal preparations are traditionally used in the treatment of cancer therapy but there may be bioactivated components that may be responsible to promote cancer. In India, the majority of population uses traditional natural preparation derived from the plant material for the treatment of various diseases, and for that reason it becomes necessary to assess the mutagenic potential or modulating action of plant extract when associated with other substances. The genotoxicity testing provides human a risk assessment. An increase in the frequency of chromosomal aberrations in peripheral blood lymphocytes is associated with an increased overall risk of cancer (Hagmar et al., 1994; Hagmar et al., 1998). The ready quantifiable nature of sister chromatid exchanges with high sensitivity for revealing toxicant-DNA interaction and the demonstrated ability of genotoxic chemicals to induce significant increase in sister chromatid exchanges in cultured cells has resulted this endpoint being used as indicator of DNA damage in blood lymphocytes of individuals exposed to genotoxic carcinogens (Albertini et al., 2000). The above

11

genotoxic end-points are well known markers of genotoxicity and any reduction in the frequency of these genotoxic end-points gives an indication of the antigenotoxicity of a particular compound or plant infusion/extract, while an increase is associated with the possibility of carcinogenesis (Albertini et al., 2000). Synthetic progestins after oral administration readily absorbed through the intestinal lining with maximum plasma concentration being reached within 30–60 min, then they undergo first pass metabolism in liver (Martelli et al., 2003). However, markedly higher blood concentrations may be reached in some clinical conditions (Martelli et al., 2003), hence the present study was performed on cultured human lymphocytes, using two different concentrations of CPA. Since the plant extract have compounds, that may enhance or reduce the genotoxic effect of a particular compound, the knowledge of a particular plant extract will contribute us to form the basis of herbal medicine (Roncada et al., 2004). Earlier in vitro and in vivo studies reveal that the plant extract from the various parts of the plant plays an important role in xenobiotic effects (Ren et al., 2001). The objective of the present work was to study the effect of C. asiatica L. extract against the genotoxic doses of CPA, on human lymphocytes in vitro. 2. Material and methods 2.1. Chemicals Cyproterone acetate (CAS No: 427-1-0, Sigma); RPMI 1640, Fetal calf serum, Phytohaemagglutinin-M, Antibiotic-antimycotic mixture (Gibco); Dimethylsulphoxide, 5Bromo-2-deoxyuridine, Colchicine (SRL, India); Giemsa stain (Merk). 2.2. Extract peparation C. asiatica L. leaves were collected from the nursery of Forest Research Institute (FRI), Dehradun (UA) and were air dried and ground to fine powder. Extraction was performed by soaking samples (30 gm of dry weight) in 300 ml of acetone for 8–10 h at 40–60 C in soxhlet’s apparatus. After filtration, the excess of solvent was removed by rotatory evaporator. The extract concentrations of 1.075 · 10 4, 2.127 · 10 4, 3.15 · 10 4 and 4.17 · 10 4 g/ml of culture medium were established (Siddique et al., 2007). 2.3. Human lymphocyte culture Duplicate peripheral blood cultures were prepared according to Carballo et al. (1993). Briefly, heparinized blood samples (0.5 ml), were obtained from healthy female donors and were placed in a sterile culture bottle containing 7 ml of RPMI-1640 medium, supplemented with fetal calf serum (1.5 ml), antibiotic–antimycotic mixture (1.0 ml)

12

Y.H. Siddique et al. / Toxicology in Vitro 22 (2008) 10–17

After 24 h, 20 lM of CPA (dissolved in dimethylsulphoxide, 5 ll/ml) treatment was given separately with 1.075 · 10 4, 2.125 · 10 4, 3.15 · 10 4 and 4.17 · 10 4 g/ml of C. asiatica L. extract respectively. Similar treatments were given with 30 lM of CPA and kept for another 48 h in an incubator. Mitotic arrest was performed by adding 0.2 ml of colchicine (0.2 lg/ml). Hypotonic treatment and fixation were performed in the same way as for the chromosomal aberration analysis. The sister chromatid exchange average was taken from an analysis of metaphase during second cycle of division (Perry and Wolff, 1974).

and phytohaemagglutinin (0.1 ml). The culture bottles were kept in an incubator at 37 C for 24 h. 2.4. Chromosomal aberration analysis After 24 h, 20 lM of CPA treatment (dissolved in dimethyl sulphoxide, 5 ll/ml) was given separately with 1.075 · 10 4, 2.125 · 10 4 3.15 · 10 4 and 4.17 · 10 4 g/ml of Centella asiatica L. extract. Similarly, 30 lM of CPA treatment was also given with the same four dosages of C. asiatica extract. The culture bottles were kept for another 48 h in an incubator at 37 C. After 47 h, 0.2 ml of colchicine (0.2 lg/ml) was added to the culture bottle. Cells were centrifuged at 800 g for 10 min. The supernatant was removed and 5 ml of prewarmed (37 C) KCl hypotonic solution (0.075 M) was added. Cells were resuspended and incubated at 37 C for 15 min. The supernatant was removed by centrifugation at 800 g for 10 min, and 5 ml of chilled fixative (methanol: glacial acetic acid; 3:1) was added. The fixative was removed by centrifugation and the procedure was repeated twice. The slides were stained in 3% Giemsa solution in phosphate buffer (pH 6.8) for 15 min. Three-hundred metaphases were examined for the occurrence of different types of abnormality. Criteria to classify the different types of aberrations were in accordance with recommendation of EHC 46 for environmental monitoring of human Population (IPCS, 1985).

2.6. Statistical analysis Student ‘t’-test was used for analysis of CAs and SCEs. Regression analysis was performed using statistica soft Inc. 3. Results Cyproterone acetate (CPA) induced a significant increase of abnormal metaphases as compared to the untreated. A significant dose dependent decrease in number of abnormal metaphase was observed when 20 and 30 lM of CPA was treated, separately, with the different dosages of C. asiatica extract, i.e. 1.075 · 10 4, 2.125 · 10 4, 3.15 · 10 4 and 4.17 · 10 4 g/ml (Table 1; Fig. 1). For sister chromatid exchange analysis, a significant increase was observed at both the dosages of CPA, i.e. 20 and 30 lM (Table 2; Fig. 2). A significant decrease in sister chromatid exchanges per cell was observed when 20 and 30 lM of CPA was treated, separately, with the different dosages of C. asiatica extract, i.e. 1.075 · 10 4, 2.125 · 10 4, 3.15 · 10 4 and

2.5. Sister chromatid exchange analysis For sister chromatid exchange analysis, bromodeoxyuridine (10 lg/ml) was added at the beginning of the culture.

Table 1 Effect of Centella asiatica L. extract on chromosomal aberrations (CA) induced by cyproterone acetate (N = 2) Treatment

Abnormal metaphases without gaps

Chromosome aberrations

Number

Mean% ± SE

Gaps number

Fragments number

CTB number

CSB number

11 16

3.67 ± 1.08a 5.33 ± 1.29a

6 10

3 7

9 14

3 5

CPA (lM) + CAE (g/ml) 20 + 1.075 · 10 4 9 12 30 + 1.075 · 10 4 20 + 2.125 · 10 4 8 30 + 2.125 · 10 4 10 6 20 + 3.15 · 10 4 30 + 3.15 · 10 4 7 20 + 4.17 · 10 4 5 30 + 4.17 · 10 4 5

3.00 ± 0.98b 4.00 ± 1.13b 2.67 ± 0.93b 3.33 ± 1.03b 2.00 ± 0.80b 2.33 ± 0.87b 1.67 ± 0.73b 1.67 ± 0.73b

4 8 3 7 2 6 2 5

2 5 1 3 – 2 – 1

7 10 5 8 2 3 2 2

2 3 1 1 1 1 – 1

Untreated CAE (g/ml) 1.075 · 10 4 2.125 · 10 4 3.15 · 10 4 4.17 · 10 4

2 3 3 4 4

0.66 ± 0.46 1.00 ± 0.57 1.00 ± 0.57 1.33 ± 0.56 1.33 ± 0.56

1 1 1 2 2

– – – – –

2 2 2 2 3

– 1 1 1 1

Negative control (DMSO, 5 ll/ml)

3

1.00 ± 0.57

2

–

2

1

CPA (lM) 20 30

CPA: Cyproterone acetate; CAE: Centella asiatica extract; DMSO: Dimethylsulphoxide; CTB: Chromatid break; CSB: Chromosome break. a P < 0.01 Significant with respect to untreated. b P < 0.05 Significant with respect to CPA treatment.

13

18 16 14 12 10 8 6 4 2

U

NC

CA E1 CA E2 CA E3 CA E4

CP A1 +C CP AE A1 1 +C CP AE A1 2 +C CP AE A1 3 +C AE 4 CP A2 CP +CAE A2 1 +C CP AE A2 2 +C CP AE A2 3 +C AE 4

0

CP A1 CP A2

No. of Abnormal metaphases without gaps

Y.H. Siddique et al. / Toxicology in Vitro 22 (2008) 10–17

Fig. 1. Effect of Centella asiatica L. extract on abnormal metaphases induced by cyproterone acetate (N = 2). CPA = Cyproterone acetate CPA1 = 20 lM; CPA2 = 30 lM CPA1 + CAE1 = 20 lM + 1.075 l10 4 g/ml CPA1 + CAE2 = 20 lM + 2.125 l10 4 g/ml CPA1 + CAE3 = 20 lM + 3.15 l10 4 g/ml CPA1 + CAE4 = 20 lM + 4.17 l10 4 g/ml

CAE = Centella asiatica extract U = Untreated CPA2 + CAE1 = 30 lM + 1.075 l10 4 g/ml CPA2 + CAE2 = 30 lM + 2.125 l10 4 g/ml CPA2 + CAE3 = 30 lM + 3.15 l10 4 g/ml CPA2 + CAE4 = 30 lM + 4.17 l10 4 g/ml

4.17 · 10 4 g/ml (Table 2; Fig. 2). Regression analysis was also performed to determine the dose effect of C. asiatica extract on 20 and 30 lM of CPA, for number of abnormal metaphases and sister chromatid exchanges. A decrease in the slope of linear regression lines was observed as the dose of the extract was increase, in each of the treatment. For

Table 2 Effect of Centella asiatica L. extract on sister chromatid exchanges (SCE) induced by cyproterone acetate (N = 2) Treatment

SCEs/Cell (Mean ± SE)

Range

CPA (lM) 20 30

6.02 ± 0.43a 8.30 ± 0.62a

2–7 2–9

CPA (lM) + CA (g/ml) 20 + 1.075 · 10 4 30 + 1.075 · 10 4 20 + 2.125 · 10 4 30 + 2.125 · 10 4 20 + 3.15 · 10 4 30 + 3.15 · 10 4 20 + 4.17 · 10 4 30 + 4.17 · 10 4

4.22 ± 0.32b 6.34 ± 0.44b 3.32 ± 0.33b 4.26 ± 0.38b 2.88 ± 0.17b 3.36 ± 0.25b 2.52 ± 0.16 3.02 ± 0.22

2–5 2–8 2–5 2–5 2–5 2–5 2–5 2–5

1.24 ± 0.11 2.12 ± 0.18 2.32 ± 0.21 2.64 ± 0.23 2.70 ± 0.26

0–5 0–5 0–5 0–5 0–5

1.82 ± 0.15

0–5

Untreated CAE (g/ml) 1.075 · 10 4 2.125 · 10 4 3.15 · 10 4 4.17 · 10 4 Negative control (DMSO, 5 ll/ml)

CPA: Cyproterone acetate; CAE: Centella asiatica extract; DMSO: Dimethylsulphoxide. a P < 0.01 Significant with respect to untreated. b P < 0.05 Significant with respect to CPA treatment.

NC = Negative Control (DMSO 5 ll/ml) CAE1 = 1.075 l10 4 g/ml CAE2 = 2.125 l10 4 g/ml CAE3 = 3.15 l10 4 g/ml CAE4 = 4.17 l10 4 g/ml

abnormal metaphases the treatment of 20 lM (F = 95.14; P < 0.001) and 30 lM (F = 273.3; P < 0.001) of CPA, with the increase in the dosages of C. asiatica extract result in the decrease in slope of the linear regression lines (Figs. 3 and 4). For sister chromatid exchange analysis, the treatment of 20 lM (F = 40.18; P < 0.002) and 30 lM (F = 15.60; P < 0.01) of CPA, with the increase in the dosages of C. asiatica extract, the decrease in slope of linear regression lines was observed (Figs. 5 and 6). 4. Discussion The results of the study reveal that the selected dosages of the plant extract were not genotoxic per se but reduced the genotoxic damage of cyproterone acetate (CPA) on human lymphocytes in vitro. In our earlier study, four doses of CPA (5, 10, 20 and 30 lM) were studied (Siddique and Afzal, 2005b). CPA was found genotoxic at 20 and 30 lM. The International Agency on Cancer (IARC), mainly on the basis of epidemiological studies classifies steroidal estrogens and estrogen progestin combinations among agents carcinogenic to humans (Group 1), progestins as possibly carcinogenic (Group 2) and androgenic anabolic steroids, as probably carcinogenic (Group 2A) (Martelli et al., 2003). Carcinogenicity to humans of sex steroids has been evaluated, and is reported that high dose of estrogen–progestin combinations can cause liver cancer to humans (IARC, 1999). In a very recent ‘‘Multi centre study’’ on oral contraceptives and liver cancer the ‘‘Project Team’’ came to the conclusion that oral contraceptives may enhance the risk of liver carcinomas (Martelli et al., 2003). CPA is not only a tumor promoting agent but also a genotoxic chemical (Joosten et al., 2004). Sister chromatid

Y.H. Siddique et al. / Toxicology in Vitro 22 (2008) 10–17 10 9 8 7 6 5 4 3 2 1

U

NC

CA E1 CA E2 CA E3 CA E4

CP A1 +C CP AE A1 1 +C CP AE A1 2 CP +CAE A1 3 +C AE 4 CP A2 CP +CAE A2 1 +C CP AE A2 2 CP +CAE A2 3 +C AE 4

0

CP A1 CP A2

Sister chromatid exchanges/cell

14

Fig. 2. Effect of Centella asiatica L. extract on sister chromatid exchanges induced by cyproterone acetate (N = 2). CPA = Cyproterone acetate CPA1 = 20 lM; CPA2 = 30 lM CPA1 + CAE1 = 20 lM + 1.075 l10 4 g/ml CPA1 + CAE2 = 20 lM + 2.125 l10 4 g/ml CPA1 + CAE3 = 20 lM + 3.15 l10 4 g/ml CPA1 + CAE4 = 20 lM + 4.17 l10 4 g/ml

CAE = Centella asiatica extract U = Untreated CPA2 + CAE1 = 30 lM + 1.075 l10 4 g/ml CPA2 + CAE2 = 30 lM + 2.125 l10 4 g/ml CPA2 + CAE3 = 30 lM + 3.15 l10 4 g/ml CPA2 + CAE4 = 30 lM + 4.17 l10 4 g/ml

NC = Negative Control (DMSO 5 ll/ml) CAE1 = 1.075 l10 4 g/ml CAE2 = 2.125 l10 4 g/ml CAE3 = 3.15 l10 4 g/ml CAE4 = 4.17 l10 4 g/ml

Y = 10.570 - 1.357 * X

Y = 14.621 - 2.327 * X

Correlation: r = -.9897

Correlation: r = -.9964 13

Number of abnormal metaphases

Number of abnormal metaphases

9.5

8.5 Regression 95% confid. 7.5

6.5

5.5

4.5 0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

Concentration of extract

12 11 Regression 95% confid.

10 9 8 7 6 5 4 0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

Concentration of extract

Fig. 3. Regression analysis for the dose effect of C. asiatica extract on abnormal metaphases induced by cyproterone acetate (20 lM). Concentration of extract is in (· 10 4 g/ml).

Fig. 4. Regression analysis for the dose effect of C. asiatica extract on abnormal metaphases induced by cyproterone acetate (30 lM). Concentration of extract is in (· 10 4 g/ml).

exchanges (SCE) have been commonly used to evaluate cytogenetic responses to chemical exposure, and an excellent dose response relationship has been established for hundred of chemicals in a wide variety of in vivo and in vitro short term experiments (Tucker and Preston, 1996). Chromosomal aberrations are changes in chromosome structure resulting from a break or an exchange of chromosomal material. Most of the chromosomal aberrations observed in the cells are lethal, but there are many corresponding aberration that are viable and cause genetic effects, either somatic or inherited (Swierenga et al., 1991). These events lead to the loss of chromosomal material at mitosis or due to the inhibition of accurate chromosome

segregation at anaphase. SCE is generally a more sensitive indicator of genotoxic effects than structural aberrations (Tucker and Preston, 1996). There is a correlation between the carcinogenicity and SCE inducing ability of large number of chemicals (Gebhart, 1981). Concerning our earlier study on CPA it was found genotoxic by generating free radicals in the test system (Siddique and Afzal, 2005b). An excess of reactive oxygen species (ROS) leads to the DNA damage. Many plant products protect against xenobiotics either by inducing detoxifying enzymes or by inhibiting oxidative enzymes (Morse and Stoner, 1993). The verification of the possible mutagenic and/or anti-mutagenic effects of medicinal plants infusion/extracts is another

Y.H. Siddique et al. / Toxicology in Vitro 22 (2008) 10–17 Y = 4.6503 - .5382 * X Correlation: r = -.9764

Sister chromatid exchanges / cell

4.4

Regression 95% confid. 4.0

3.6

3.2

2.8

2.4 0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

Concentration of extract

Fig. 5. Regression analysis for the dose effect of C. asiatica extract on sister chromatid exchanges induced by cyproterone acetate (20 lM). Concentration of extract is in (· 10 4 g/ml).

Y = 7.0169 - 1.055 * X Correlation: r = -.9415

Sister chromatid exchanges / cell

7.0 Regression 95% confid.

6.5 6.0 5.5 5.0 4.5

15

cyclophosphamide, the extract of C. asiatica reduced the genotoxic damage this is due to the possible prevention of metabolic activation of cyclophosphamide by the extract (Siddique et al., 2007). The compounds present in the extract may also scavenge electrophiles/nucleophiles (Maurich et al., 2004). In the present study, the extract may possibly scavenge free radicals generated by CPA via nucleophilic reaction (Siddique and Afzal, 2005b) and reduced the genotoxic damage. The compounds may also enhance the DNA repair system or DNA synthesis or even may prevent the bio-activation of certain chemicals (Kuroda et al., 1992). The treatment of extract reduced the frequency of SCE and chromosomal aberrations in the test system, thereby indicating the possibility of reducing the chances of carcinogenesis during the CPA therapy in patients. The antigenotoxic potential of the plant extracts have been attributed to their total phenolic content (Maurich et al., 2004). Medicinal herbs contain complex mixtures of thousand of compounds that can exert their antioxidant and free radical scavenging effect either separately or in synergistic ways (Romero-Jimenez et al., 2005). Identification and characterization of these active principles in the plant extract may lead to the strategies to reduce the risk for developing cancer in humans (Dearfield et al., 2002). The identification and characterization of the compounds present in the C. asiatica extract to determine their particular functions will be the part of our future study, however at present it can be concluded from the study that C. asiatica extract has the potential to reduced the genotoxic damage induced by CPA in cultured human lymphocytes.

4.0 3.5

Acknowledgements

3.0 2.5 0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

Concentration of extract

Fig. 6. Regression analysis for the dose effect of C. asiatica extract on sister chromatid exchanges induced by cyproterone acetate (30 lM). Concentration of extract is in (· 10 4 g/ml).

Thanks are due to the CSIR, New Delhi for awarding the Fellowship No. 9/112(355)/2003-EMR to the author (YHS) and to the Chairman, Department of Zoology, A.M.U., Aligarh, U.P., for laboratory facilities. References

important factor in studies. Such effects have been elucidated in some plant species by using various test systems (Roncada et al., 2004). Some plants may possess substances that can modulate the genotoxicity of other compounds. The data obtained in the present study suggests that the compound present in the extract of C. asiatica, are not mutagenic on their own or when associated with the CPA. The extract of C. asiatica is capable of reducing the genotoxic effect of CPA. The protective effect observed in the present study, i.e. significant reduction in the frequency of cells with chromosomal damage and sister chromatid exchanges may be due to the direct action of the compounds present in the extract on CPA by inactivating it enzymatically or chemically. The genotoxic effects of CPA in liver cells have been attributed to the bio-activation of CPA by hydroxyl steroid sulfotransferase (HST) to reactive metabolites (Kasper and Mueller, 1999). In our earlier study with

Ahmad, S., Hoda, A., Afzal, M., 2002. Additive action of vitamin C and E against hydrocortisone induced genotoxicity in human lymphocytes chromosomes. International Journal of Vitamin and Nutrition Research 72, 204–209. Albertini, R.J., Anderson, D., Douglas, G.R., Hagmar, L., Heminiki, K., Merelo, F., Natrajan, A.T., Norppa, H., Shuker, D.E.G., Tice, R., Walters, M.D., Aitio, A., 2000. IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans. Mutation Research 463, 111–172. Babu, T.D., Kuttar, G., Padikkla, J., 1995. Cytotoxic and antitumor properties of certain taxa of umbelliferae with special reference to Centella asiatica (L) Urban. Journal of Ethnopharmacology 48, 53–57. Carballo, M.A., Alvarez, S., Boveris, S., 1993. Cellular stress by light and Rose Bengal in human lymphocytes. Mutation Research 288, 215–222. Dastur, J.F., 1962. Medicinal Plants of India and Pakistan. D.B. Taraporevala Sons & Co., Bombay. Dearfield, K.L., Cimino, M.C., Mc Carroll, N.E., Mauer, I., Valcovic, L.R., 2002. Genotoxicity risk assessment: a proposed classification strategy. Mutation Research 521, 121–135.

16

Y.H. Siddique et al. / Toxicology in Vitro 22 (2008) 10–17

Deml, E., Schwarz, L.R., Oesterle, D., 1993. Initiation of enzyme altered foci by the synthetic steroid cyproterone acetate in rat liver foci bioassay. Carcinogenesis 14, 1229–1231. Dutta, T., Basu, U.P., 1968. Crude extract of Centella asiatica and products derived from its glycosides as oral antifertility agents. Indian Journal of Experimental Biology 6, 181–182. Gebhart, E., 1981. Sister chromatid exchange (SCE) and structural chromosome aberrations in mutagenicity testing. Human Genetics 58, 235–254. Guo, J.S., Cheng, C.L., Koo, M.W., 2004. Inhibitory effects of Centella asiatica water extract and asiaticoside on inducible nitric oxide synthase during gastric ulcer healing in rats. Planta Medica 70, 1150–1154. Gupta, R., Flora, S.J., 2006. Effect of Centella asiatica on arsenic induced oxidative stress and mental distribution in rats. Journal of Applied Toxicology 26, 213–222. Hagmar, L., Brogger, A., Hansteen, I.L., Heims, S., Hogstedt, B., Knudsen, L., Lambert, B., Linnainmaa, K., Mitelman, F., Nordenson, I., Reuterwall, C., Salomaa, S., Skerfving, S., Sorsa, M., 1994. Cancer risk in humans predicted by increased level of chromosomal aberration in human lymphocytes: Nordic study group on the health risk of chromosome damage. Cancer Research 54, 2919– 2922. Hagmar, L., Bonassi, S., Stromberg, U., Brogger, A., Knudson, J.E., Norppa, H., Reuterwall, C., 1998. Chromosomal aberrations in human lymphocytes predict human cancer: A report from the European study group on cytogenetic biomarkers and health (ESCH). Cancer Research 58, 4117–4121. IARC. 1999. International Agency for Research on Cancer, Oral Contraceptives, Combined (Group 1) vol. 72, p. 49. IARC. 1999. Monographs on the Hormonal Contraceptives, Progestogens Only (Group 2B). Inamdar, P.K., Yeole, K.D., Cibogare, A.B., Desouza, N.J., 1996. Determination of biologically active constituents in Centella asiatica. Journal of Chromatography 742, 127–130. IPCS. 1985. International Programme on Chemical Safety: Environmental Health Criteria 46. Guidelines for the study of genetic effects in human populations, WHO Geneva, pp. 25–54. Joosten, H.F.P., van Acker, F.A.A., van de Dobbelsten, Horbach, D.J., Kranjc, E.I., 2004. Genotoxicity of hormonal steroids. Toxicology Letters 151, 113–134. Kasper, P., Mueller, L., 1999. Sex specific induction of apoptosis by cyproterone acetate in primary rat hepatocytes. Carcinogenesis 20, 2185–2188. Kasper, P., Tegethoff, K., Mueller, L., 1995. In vitro mutagenicity studies on cyproterone studies on cyproterone acetate using female rat hepatocytes for metabolic activation as indicator cells. Carcinogenesis 16, 2309–2314. Kuroda, Y., Jain, A.K., Tezuka, H., Kada, T., 1992. Antimutagenicity in cultured mammalian cells. Mutation Research 267, 201–209. Lu, L., Ying, K., Wei, S., Fang, Y., Liu, Y., Liu, H., Ma, L., Mao, Y., 2004. Asiaticoside induction for cell cycle progression, proliferation and collagen synthesis in human dermal fibroblast. International Journal of Dermatology 43, 801–807. Martelli, A., Campart, G.B., Ghia, M., Allavena, A., Mereto, E., Brambilla, G., 1996. Induction of micronuclei and initiation of enzyme altered foci in the liver of female rats treated with cyproterone acetate, chlormadinone acetate or megestrol acetate. Carcinogenesis 17, 551– 554. Martelli, A., Mattioli, F., Angiola, M., Reimann, R., Brambilla, G., 2003. Species, sex and interindividual differences in RNA repair induced by nine sex steroids in primary cultures of rat and human hepatocytes. Mutation Research 536, 69–78. Maurich, T., Pistelli, L., Turchi, G., 2004. Anti-clastogenic activity of two structurally related pterocarpans purified from Bituminaria bituminosa in cultured human lymphocytes. Mutation Research 561, 75–81. Morse, M.A., Stoner, G.D., 1993. Cancer chemoprevention: Principles and prospects. Carcinogenesis 14, 1737–1746.

Park, B.C., Bosire, K.O., Lee, E.S., Lee, Y.S., Kim, J.A., 2005. Asiatic acid induces apoptosis in SK-MEL-2 human melanoma cell. Cancer Letters 218, 81–90. Perry, P., Wolff, S., 1974. New Giemsa methods for differential staining of sister chromatids. Nature 261, 156–158. Punuree, K., Wild, C.P., Kasinrerk, W., Vinitketkumnuen, U., 2005. Immunodulatory activities of Centella asiatica and Rhimachanthus nasutus extracts. Asian Pacific Journal of Cancer Prevention 6, 396–400. Reimann, R., Kalweit, S., Lang, R., 1996. Studies for a genotoxic potential of some endogenous and exogenous sex steroid. II. communication: Examination for the induction of cytogenetic damage using the chromosomal aberration assay on human lymphocytes in vitro and the mouse bone marrow micronucleus test in vivo. Environmental and Molecular Mutagenesis 28, 133–144. Ren, H., Endo, H., Hayashi, T., 2001. The superiority of organically cultivated vegetables to general one regarding anti-mutagenic activities. Mutation Research 496, 83–88. Romero-Jimenez, M., Sanchez, J.C., Analla, M., Serrano, A.M., Moraga, A.A., 2005. Genotoxicity and antigenotoxicity of some traditional medicinal herbs. Mutation Research 585, 147–155. Roncada, T., Vicentini, V.E.P., Mantovani, M.S., 2004. Possible modulating actions of plants extracts on the chromosome breaking activity of MMC and Ara C in human lymphocytes in vitro. Toxicology in vitro 18, 617–622. Sampson, J.H., Raman, A., Karlsen, G., Navsaria, H., Leigh, I.M., 2001. In vitro keratinocyte antiproliferant effect of Centella asiatica extract and triterpinoid saponin. Phytomedicine 8, 230–235. Sharma, J., Sharma, R., 2002. Radioprotection of Swiss albino mouse by Centella asiatica extract. Phytotherapy Research 16, 785–786. Shukla, A., Rasik, A.M., Dhawan, D.N., 1999. Asiaticoside induced elevation of antioxidant levels in healing wounds. Phytotherapy Research 13, 50–54. Siddique, Y.H., Afzal, M., 2004a. Evaluation of genotoxic potential of synthetic progestin chlormadinone acetate. Toxicology Letters 153, 221–225. Siddique, Y.H., Afzal, M., 2004b. Evaluation of genotoxic potential of synthetic progestin ethynodiol diacetate in human lymphocytes in vitro. Current Science 86, 1161–1168. Siddique, Y.H., Afzal, M., 2005a. Protective role of allicin and L-ascorbic acid against the genotoxic damage induced by chlormadinone acetate in cultured human lymphocytes. Indian Journal of Experimental Biology 43, 769–772. Siddique, Y.H., Afzal, M., 2005b. Genotoxic potential of cyproterone acetate: a possible role of reactive oxygen species. Toxicology in vitro 19, 63–68. Siddique, Y.H., Beg, T., Afzal, M., 2005. Antigenotoxic effects of ascorbic acid against megestrol acetate induced genotoxicity in mice. Human and Experimental Toxicology 24, 121–127. Siddique, Y.H., Beg, T., Afzal, M., 2006a. Protective effect of nordihydroguaiaretic acid (NDGA) against norgestrel induced genotoxic damage. Toxicology in vitro 20, 227–233. Siddique, Y.H., Ara, G., Beg, T., Afzal, M., 2006b. Effect of vitamin C on cyproterone acetate induced genotoxic damage in mice. Research Journal of Biological Sciences 1, 69–73. Siddique, Y.H., Ara, G., Beg, T., Faisal, M., Ahmad, M., Afzal, M., 2007. Protective role of Centella asiatica L. extract against methyl methanesulphonate and cyclophosphamide induced genotoxic damage in cultured human lymphocytes. Recent Progress in Medicinal Plants 19, 369–381. Siddique, Y.H., Ara, G., Beg, T., Afzal, M. (in press). Antigenotoxic effect of nordihydroguaiaretic acid against chlormadinone acetate – induced genotoxicity in mice bone marrow cells. Journal of Natural Medicines. Swierenga, S.H.H., Heddle, J.A., Sigal, E.A., Gilman, J.P.W., Brillinger, R.L., Douglas, G.R., Nestmann, E.R., 1991. Recommended protocols on a survey of current practice in genotoxicity testing laboratories, IV. Chromosome aberrations and sister chromatid exchanges in Chinese Hamster Ovary V79, Chinese Hamster Lung and human lymphocyte cultures. Mutation Research 246, 301–322.

Y.H. Siddique et al. / Toxicology in Vitro 22 (2008) 10–17 Tucker, J.D., Preston, R.J., 1996. Chromosomes aberrations, micronuclei aneuploidy, sister chromatid exchanges and cancer risk assessment. Mutation Research 365, 147–159. Yoshida, M., Fuchigami, M., Nago, T., Okabe, H., Matsunaga, K., Takata, J., Karube, J., Tsuchihashi, R., Kimjo, J., Mihashi, K., Fujioka, T., 2005. Antiproliferative constituents from umbelliferae

17

plants VII. Active triterpene and rosmarinic acid from Centella asiatica. Biology and Pharmacology Bulletin 28, 173–175. Zainol, M.K., Hamid, A.A., Yusof, S., Muse, R., 2003. Antioxidative activity and total phenolic compounds of leaf, root and peephole of four accessions of Centella asiatica (L.) urban. Food Chemistry 81, 575–581.